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Cellular Microbiology: Pathogen-Host Cell Molecular Interactions

PerR Controls Oxidative Stress Resistance and Iron Storage Proteins and Is Required for Virulence in Staphylococcus aureus

Malcolm J. Horsburgh, Mark O. Clements, Howard Crossley, Eileen Ingham, Simon J. Foster
Malcolm J. Horsburgh
Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, 1 and
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Mark O. Clements
Microbiology and Tumour Biology Centre, Karolinska Institute, 17177 Stockholm, Sweden 2
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Howard Crossley
Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, 1 and
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Eileen Ingham
Department of Microbiology, University of Leeds, Leeds, 3 England, and
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Simon J. Foster
Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, 1 and
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DOI: 10.1128/IAI.69.6.3744-3754.2001
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  • Fig. 1.
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    Fig. 1.

    (A) Effect of H2O2 (7.5 mM) on washed, exponential-phase CLR-grown cells of 8325-4 (wild type) (▪), MJH001 (perR) (○), and ST16 (katA) (▴) and 8325-4 (wild type) grown in CLR with 20 μM manganese chloride (■). (B) Catalase activity gel. (C) Total catalase activity of washed, lysed, stationary-phase cells after 24 h of growth in CLR medium (open bars), CLR with 20 μM manganese chloride (gray bars), or CLR with 20 μM iron sulfate (black bars).

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    Fig. 2.

    Growth of 8325-4 (wild-type) (▪), MJH001 (perR) (▵,) and ST16 (katA) (○) strains in CL with no added metal ions except magnesium (A), CL with 20 μM iron sulfate (B), CLR (C), and CL with 20 μM manganese chloride (D). All cultures were inoculated at an OD600 of 0.002.

  • Fig. 3.
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    Fig. 3.

    Alignment of the putative PerR boxes identified in the incomplete S. aureus genome databases. Boxes were identified as described previously (37). An S. aureus consensus sequence was compiled from all of the sequences identified and compared to the B. subtilisconsensus (19).

  • Fig. 4.
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    Fig. 4.

    (A) Mapping of the 5′ end of the perR,katA, mrgA, ahpC, andfur mRNA by primer extension. RNA was isolated from exponential cultures of 8325-4 (wild type) grown in CLR (OD600 of 1.0), and 100 μg of RNA was used for each reaction. The potential transcription start sites are marked with arrows. Lanes G, T, A, and C show the dideoxy sequencing ladder obtained with the same primer used for primer extension (x). (B) Sequence of the identified promoter regions. Potential −35 and −10 regions and transcriptional start sites are underlined. PerR boxes are in boldface.

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    Fig. 5.

    Analysis of transcription from promoter-lacZ fusions during growth in CLR medium. 8325-4 (wild type) containing the fusion indicated was grown in CLR medium (▴), CLR with 20 μM manganese (⋄), and CLR with 20 μM iron sulfate (○). Transcription of the lacZ fusions in MJH001 (perR) (▪) is shown on each graph. Samples were removed at the times indicated and sampled for β-galactosidase activity. The strains grew with slightly different growth rates as indicated in Fig. 2, but for reasons of clarity only the changes in β-galactosidase activity are shown. The lacZfusions shown are MJH002 (ahpC-lacZ), MJH003 (bcp-lacZ), MJH004 (ftn-lacZ), MJH005 (fur-lacZ), MJH006 (katA-lacZ), MJH007 (mrgA-lacZ), MJH008 (perR-lacZ), and MJH009 (trxB-lacZ), and these same fusions in theperR mutant background are MJH102, MJH103, MJH104, MJH105, MJH106, MJH107, MJH108, and MJH109, respectively.

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    Fig. 6.

    Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins from wild-type and MJH001 (perR) strains after growth to stationary phase in CLR at 37°C. The AhpC, Ftn, and MrgA proteins identified by N-terminal sequencing are shown.

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    Fig. 7.

    Starvation survival of 8325-4 (wild type) (■), MJH001 (perR) (▴), and ST16 (katA) (●) after growth and incubation in CDM containing 0.1% (wt/vol) glucose (glucose limiting). Samples were aseptically removed at the times indicated, and viability was assessed by dilution and counting on BHI agar.

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    Fig. 8.

    Pathogenicity of S. aureus strains in a murine skin abscess model of infection. Approximately 108CFU of each strain was inoculated subcutaneously into 6- to 8-week-old BALB/c mice; the strains used were 8325-4 (wild type) (n = 10), MJH001 (perR) (n = 10), and ST16 (katA) (n = 16). Seven days after infection, mice were euthanized, lesions were removed and homogenized, and viable bacteria were counted after dilution and growth on BHI agar plates. The mean percent recovery of each strain and the Studentt test P values are as follows, respectively: 8325-4 (wild type), 143%; MJH001 (perR), 10.5% and P < 0.005; and ST16 (katA), 86.3% and P = 0.124.

Tables

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  • Table 1.

    Strains, plasmids, and primers used in this study

    Strain, plasmid, or primerGenotype, description, or sequenceaReference or source
    Strains
     E. coli
      DH5αφ80 Δ(lacZ)M15 Δ(argF-lac)U169 endA1 recA1 hsdR17 (rK − mK +) deoR thi-1 supE44 gyrA96 relA1 56
     S. aureus
      8325-4Wild-type strain cured of prophagesLab stock
      RN4220Restriction-deficient transformation recipientLab stock
      MJH001 perR::kan This study
      MJH002 ahpC::pAZ106ahpC + This study
      MJH003 bcp::pAZ106bcp + This study
      MJH004 ftn::pAZ106ftn + This study
      MJH005 fur::pAZ106fur + This study
      MJH006 katA::pAZ106katA + This study
      MJH007 mrgA::pAZ106mrgA + This study
      MJH008 perR::pAZ106perR + This study
      MJH009 trxB::pAZ106trxB + This study
      ST16 katA::Tn917-LTV1This study
      SPW1 sodA::Tn917-LTV1 65
      MJH102 perR::kan ahpC::pAZ106 ahpC + This study
      MJH103 perR::kan bcp::pAZ106 bcp + This study
      MJH104 perR::kan ftn::pAZ106 ftn + This study
      MJH105 perR::kan fur::pAZ106 fur + This study
      MJH106 perR::kan katA::pAZ106 katA + This study
      MJH107 perR::kan mrgA::pAZ106 mrgA + This study
      MJH108 perR::kan perR::pAZ106This study
      MJH109 perR::kan trxB::pAZ106 trxB + This study
      MJH408 perR::kan pMAL34 (perR +)This study
      MJH4188325-4 pMAL34 (perR +)This study
    Plasmids
     pAZ106Promoterless lacZ erminsertion vector 16
     pGem3Zf(+)General cloning vectorPromega
     pAUL-ATemperature-sensitiveerm integrational shuttle vector 15
     pCU1 E. coli-S. aureus cat shuttle vector 7
     pMAL5pGem3Zf(+) containing a 2-kb perR fragment with engineered ClaI siteThis study
     pMAL7pMAL5 containing a kancassette in ClaI site.This study
     pMAL83.5-kbBamHI-HindIIIperR::kan fragment from pMAL7 in pAUL-AThis study
     pMAL91.08-kb OL7–OL11 perRpromoter-containing fragment in pAZ106This study
     pMAL100.6-kb OL13–OL14 fur promoter-contaiing fragment in pAZ106This study
     pMAL111.2-kb OL15–OL16katA promoter-containing fragment in pAZ106This study
     pMAL121.2-kb OL17–OL18 ahpCF promoter-containing fragment in pAZ106This study
     pMAL131.1-kb OL19–OL20mrgA promoter-containing fragment in pAZ106This study
     pMAL280.6-kb OL53–OL54 bcp promoter-containing fragment in pAZ106This study
     pMAL291.4-kb OL60–OL61ftn promoter-containing fragment in pAZ106This study
     pMAL301.4-kb OL62–OL63 trxB promoter-containing fragment in pAZ106This study
     pMAL340.9-kb OL82–OL83perR gene-containing fragment in pCU1This study
    Primers
     OL7AATTGGATCCCATGGTTTGCAACGGGTGThis study
     OL8CCGGAAGCTTATCCTGAGCCAGGATCAAACTCTCCATThis study
     OL9GAATCAATTGCATCGATTGCGACAAGCAGGCGThis study
     OL10CGCCTCCTTGTCGCAATCGATGCAATTGATTCThis study
     OL11CCAGAATTCGAATCGACTTGATGAGTCTCCATATGThis study
     OL12AATTGGATCCTGTAATGGTTTGCCACTTTGCAGThis study
     OL13CCAGAATTCAGTAGCTTCGCGTTGTGGCGTTAGCThis study
     OL14AATTGGATCCATTACCAAACGGTGAAACGTCThis study
     OL15CCAGAATTCAACGCAGCTTGTTCAACATCCThis study
     OL16AATTGGATCCGACCACAATGCCCAATACAACCThis study
     OL17CCAGAATTCTGAATGTACCACGTTGAGCTAACThis study
     OL18AATTGGATCCATGGTAAGCGTGGCTTGGCTGCThis study
     OL19CCAGAATTCTACATCATCGCCAGCATTACCThis study
     OL20AATTGGATCCATGGCAATTTCGTGTCGCGAGGThis study
     OL53GGTTGGATCCAATTTCTAATTCAGTCGGTGTACCThis study
     OL54CCAGAATTCTAAATTGTCTCTAAAGTCACAAGCThis study
     OL60AGAAGGATCCGCGTTATAAGCGTTAAAGTCACThis study
     OL61AGCAGAATTCTGCATGTGCACCTCTGTCGThis study
     OL62AGAAGGATCCAGAACTGATTACGATTGGTAGThis study
     OL63AGCAGAATTCCTGCCAAATACTTTCCGTCATCThis study
     OL82AGAGGATCCACAGCGCATATAACTGGTAATGThis study
     OL83CCAGAATTCCTTATACTCACTTTATGGATAGThis study
     PEX2AGCTCCATGGTCTGACGCTCAGTGGAACGAAAACTCThis study
     PEX3CAGTAGCTTCGCGTTGTGGCGTTAGCThis study
     PEX4GGTGTAATTCTTACGCCTGCTTGTCGCThis study
     PEX5TCTCGGTCTGATACTGGATGCCThis study
     PEX6TTACAACATCTTGTTGATTACTCThis study
     Tn1GGAACGCCGTCTACTTACAAGCAGCThis study
     Tn2CTCACAATAGAGAGATGTCACCGTCThis study
    • ↵a In primer sequences, the restriction sites are underlined.

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PerR Controls Oxidative Stress Resistance and Iron Storage Proteins and Is Required for Virulence in Staphylococcus aureus
Malcolm J. Horsburgh, Mark O. Clements, Howard Crossley, Eileen Ingham, Simon J. Foster
Infection and Immunity Jun 2001, 69 (6) 3744-3754; DOI: 10.1128/IAI.69.6.3744-3754.2001

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PerR Controls Oxidative Stress Resistance and Iron Storage Proteins and Is Required for Virulence in Staphylococcus aureus
Malcolm J. Horsburgh, Mark O. Clements, Howard Crossley, Eileen Ingham, Simon J. Foster
Infection and Immunity Jun 2001, 69 (6) 3744-3754; DOI: 10.1128/IAI.69.6.3744-3754.2001
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